1. Field of the Invention
The present invention relates to molecular detection, and, more specifically, to methods and systems for quantitative multiplexed detection of one or more target molecules.
2. Background of the Invention
Biochemical assays are generally used in research, clinical, environmental, and industrial settings to detect or quantify the presence or amount of certain gene sequences, antigens, diseases, or pathogens. The assays are often used to identify organisms such as parasites, fungi, bacteria, or viruses present in a host organism or sample. Under certain conditions assays may provide a measure of quantification which may be used to calculate the extent of infection or disease and to monitor the state of a disease over time. In general, biochemical assays typically detect antigens (immunoassays) or nucleic acids (nucleic acid-based or molecular assays) extracted from samples derived from research, clinical, environmental or industrial sources.
There is an increasing demand for assays for the quantitative identification of multiple pathogens across a broad range of disciplines, including homeland security, food safety, and medical diagnostics. While there is existing technology for multiplex quantitative assays, these technologies are unable to provide adequate quantitative identification of multiple pathogens. For example, microarrays provide satisfactory levels of multiplexing for the identification of DNA but provide little or no quantitative information. On the other hand, real-time PCR provides high-quality quantitative information but the level of multiplexing is limited.
Real-time polymerase chain reaction (“PCR”), also known as quantitative real time polymerase chain reaction (“qrt-PCR”) among other designations, is a molecular biology tool used to simultaneously amplify a target DNA molecule using the well-known PCR process while quantifying the target DNA either as an absolute amount or a relative amount compared to another input. With standard PCR, the product is detected following completion of the reaction. To perform qrt-PCR, in contrast, the user amplifies the target DNA molecule much the same way, but detects the targeted DNA molecule in real time as the polymerase chain reaction progresses. Some of the most common methods used to detect the qrt-PCR product in real time are to utilize a non-specific fluorescent dye that incorporates into a double-stranded DNA product, or to use a sequence specific probe labeled with a fluorescent reporter that will only fluoresce when the probe hybridizes with the target sequence. If these methods are employed, for example, additional equipment such as a fluorescence detector will be required.
Sequence-specific probe methods require the use of a probe having a nucleotide base sequence that is substantially complementary to the targeted sequence or, alternatively, its amplicon. Under selective assay conditions, the probe will hybridize to the targeted sequence or its amplicon in a manner permitting a practitioner to detect the presence of the targeted sequence in a sample. Effective probes are designed to prevent nonspecific hybridization with any nucleic acid sequence that will interfere with detecting the presence of the targeted sequence. Probes and/or the amplicons may include a label capable of detection, where the label is, for example, a radiolabel, fluorescent dye, biotin, enzyme, electrochemical or chemiluminescent compound.
To quantify the qrt-PCR product, the detected fluorescence is plotted on a logarithmic scale against the cycle number. The amount of target in the pending reaction can then be determined by comparing the experimental results to standard results obtained using known amounts of product. This is, however, just one of the ways that the qrt-PCR product can be quantified.
Quantitative real-time PCR has numerous applications, particularly in the study of molecular biology. One particular use of qrt-PCR is to obtain quantitative information about pathogens in a sample. For quantitation a real-time measurement of fluorescent intensity, or real-time measurement of another parameter indicating an increased concentration of amplicons during an amplification reaction, is necessary. To differentiate multiple targets, particular primers with specific probes must be designed for each target. Or, in a less desirable case, only the primers need be designed and a dye such as SYBR Green applied to the reaction to indicate growing concentration of amplicons. For example, if there are 10 potential targets then typically 10 different probes are needed. The current state-of-the-art for detector/probe combinations allows for multiplexing of up to approximately 4 or 5 targets simultaneously. Most real-time PCR systems, however, are equipped only with two detectors for multiplexing detection. Using such a system, 6 separate PCR reactions are necessary to differentiate 6 hepatitis C virus (“HCV”) genotypes.
A DNA microarray is a tool used to detect the presence of a target nucleic acid sequence in a sample resulting from hybridization of the target nucleic acid in the sample to a complementary DNA sequence on the microarray. The microarray itself is a collection of up to thousands of DNA spots—called probes (or reporters)—attached to a surface (e.g., a surface of the microarray itself or a secondary surface such as beads). Microarrays are commonly formed on microscope slides or other relatively small surfaces that can be easily read by the microarray reader. The DNA probes can range from a very short or very long segment of DNA, and often comprise a short oligo, a gene, a segment of a gene, or a non-coding segment of DNA, among many other types of sequences.
To use the microarray, the nucleic acid of interest is obtained, purified, amplified, and labeled, typically either fluorescently or with a radiolabel. The labeled nucleic acid is then washed over the DNA spots, and if the nucleic acid of interest is actually present, it will hybridize to its complementary probe on the microarray. Unbound or non-specific bound nucleic acid is washed away, leaving only bound target behind. The labeled bound target will thus generate a signal at each DNA spot where it is bound, and a microarray reader can detect the fluorescent signal while determining the identity of the probe and target based on their known location on the microarray.
One of the advantages of DNA microarray technology is the multiplexing ability of the array. Since a single array can contain up to many thousands of different probes, a single microarray experiment can perform thousands of genetic tests simultaneously. Microarrays are commonly used to: (i) analyze gene expression; (ii) identify organisms; (iii) detect single nucleotide polymorphisms (“SNPs”); and (iv) compare genomes in closely related organisms, among many other well-known uses.
Accordingly, microarray technology is often used for the multiplexed identification of DNA. However, microarrays provide very little or inaccurate quantitative information about the identified DNA. There is therefore a continued need in the art for improved methods and systems for combining quantitation with multiplexed detection.